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Microbial xyloglucan Endotransglycosylase (XET)

A technology of xyloglucan and production method, which is applied in the field of microbial xyloglucan endotransglycosylase, and can solve problems such as XET that have not been reported

Inactive Publication Date: 2004-07-07
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To our knowledge, no XET derived from microorganisms has been reported

Method used

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  • Microbial xyloglucan Endotransglycosylase (XET)
  • Microbial xyloglucan Endotransglycosylase (XET)

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0021] Preparation of labeled oligosaccharides

[0022] The reducing oligosaccharide 4-O-[4-O-[4-O-[6-O-α-D-xylopyranose-β-D-glucopyranose]-6-O-(2-O -β-D-galactopyranose)-α-D-xylopyranose-β-D-glucopyranose]-6-O-(2-O-β-D-galactopyranose)-α- D-xylopyranose-β-D-glucopyranose]-D-glucose ('XLLG') (1 g) was dissolved in 25 ml containing 1 g of sodium cyanoborohydride (NaCNBH 3 ) in a saturated aqueous ammonium bicarbonate solution at 25°C in a dark place for 7 days for reductive amination. Ammonium bicarbonate is removed by drying and the (ninhydrin-reactive) aminated derivative of XLLG is purified (eg by gel filtration or cation exchange chromatography). The product should be an oligosaccharide-1-amino-1-deoxypolyhydric alcohol (deoxyalditol), a derivative in which the reducing terminal D-glucose moiety of XLLG is replaced by 1-amino-1-deoxy-D-glucitol .

[0023] Dissolve oligosaccharide-1-amino-1-deoxypolyhydric alcohol (50mg) in 3ml 3% borax (disodium tetraborate; pH=9.0-9....

Embodiment 1

[0191] Embodiment 1: screening XET positive bacterial strain

[0192] culture medium

[0193] PD agar: 39g potato dextrose agar, DIFCO0013; add deionized water to 1000ml, autoclave at 121°C for 15-20 minutes.

[0194] YPG agar: 4g yeast extract (DIFCO0127), 1g KH 2 PO 4 (Merck4873), 0.5g MgSO 4 ·7H 2 O (Merck5886), 15g glucose (Roquette101-0441), 20g agar (Merck1614), deionized water to make up to 1000ml, 121 high pressure steam sterilization for 15-20 minutes.

[0195] MEA: 20g malt extract powder (DIFC00186), 1g peptone (DIFC00118), 20g glucose (Roquette France1010441), 20g agar (Merck1614), make up to 1000ml with deionized water, and autoclave at 121°C for 15 minutes.

[0196] Medium A (per bottle): 30g wheat bran, 45ml of the following solution: 10g rofec (Roquette 101-0441), 10g NH 4 NO 3 (Merck1187), 10g KH 2 PO 4 (Merck4873), 40g Solcafloc (Dicacel, available from Dicalite-Europe-Nord, 9000 Gent, Belgium), 0.75g MgSO 4 ·7H 2 O (Merck 5886), 15g CaCO 3 , make...

Embodiment 2

[0307] Example 2: Purification and identification of Dichotomocladium hesseltinei XET

[0308] Dichotomocladium hesseltinei (CBS164.61) was inoculated on 15 PDA agar slants, and cultured at 26° C. for 7 days. The bacterial cells were washed with about 250 ml of sterile distilled water containing 0.1% Tween80, and used to inoculate 80 shake flasks containing medium B (2-3 ml / bottle). The shake flask was cultured at 26° C. with shaking at 200 rpm for 5 days, and after that, the culture solution was centrifuged at 4000 rpm for 15 minutes.

[0309] Purify the supernatant containing xyloglucan endotransglycosylase (XET) as follows:

[0310] A filter aid was added to the culture solution filtered through the filter cloth, and the solution was further filtered with a Chua depth filter disc to obtain a clear solution. The pH of the filtrate was adjusted to pH 8.0, and the filtrate was diluted with deionized water to achieve the same conductivity as 20 mM Tris / HCl (pH 8.0).

[0311] T...

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PUM

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Abstract

It has been found by a screening assay that XET activity is produced by an overwhelming array of phylogenetically dispersed microorganisms. Accordingly, the present invention relates to a xyloglucan endotransglycosylase preparation which is producible by cultivation of a microorganism expressing an XET.

Description

field of invention [0001] The present invention relates to microbial xyloglucan endotransglycosylase (xyloglucanendotransglycosylase, XET), its production method and application. Background of the invention [0002] Xyloglucan endotransglycosylase (XET) is an enzyme known from plants. To our knowledge, no XETs derived from microorganisms have been reported. [0003] Stephen C.Fry et al. Journal of Biochemistry (1992, 282, pp. 821-828) suggest that XET is responsible for cleaving and re-attaching intermicrofibrillar xyloglucan chains, and thus XET can cause cell wall relaxation required for plant cell expansion. [0004] XETs have been proposed for regulating plant morphology (see pages 21, 27-28 in EP562836). [0005] It is believed that XET is present in all plants, especially all terrestrial plants. XET has been extracted from dicotyledonous plants, monocotyledonous plants, specifically gramineous monocotyledonous plants and liliaceae monocotyledonous plants, liverwort...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/19C12N9/10C12N9/24C12N9/42C12R1/645C12R1/69C12S3/04
CPCC12N9/1051
Inventor R·I·尼尔森
Owner NOVO NORDISK AS
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